alpha-synuclein and geldanamycin

Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of alpha-synuclein, a molecule linked to Parkinson's Disease, may lead to proteasomal dysfunction. Using PC12 cells stably expressing wild-type or mutant alpha-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of alpha-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of alpha-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of alpha-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived alpha-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates.

Topics: Alanine; alpha-Synuclein; Ammonium Chloride; Animals; Benzoquinones; Cerebral Cortex; Chromatography, Gel; Coloring Agents; Congo Red; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; PC12 Cells; Proteasome Endopeptidase Complex; Protein Folding; Rats; Transfection; Tyrosine

Parkinson's disease is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of the protein alpha-synuclein into aggregates called Lewy bodies and Lewy neurites. Parkinson's disease can be modeled in Drosophila where directed expression of alpha-synuclein induces compromise of dopaminergic neurons and the formation of Lewy body-like aggregates. The molecular chaperone Hsp70 protects cells from the deleterious effects of alpha-synuclein, indicating a potential therapeutic approach to enhance neuron survival in Parkinson's disease. We have now investigated the molecular mechanisms by which the drug geldanamycin protects neurons against alpha-synuclein toxicity. Our studies show that geldanamycin sensitizes the stress response within normal physiological parameters to enhance chaperone activation, offering protection against alpha-synuclein neurotoxicity. Further, geldanamycin uncouples neuronal toxicity from Lewy body and Lewy neurite formation such that dopaminergic neurons are protected from the effects of alpha-synuclein expression despite the continued presence of (and even increase in) inclusion pathology. These studies indicate that compounds that modulate the stress response are a promising approach to treat Parkinson's disease.

Topics: alpha-Synuclein; Animals; Benzoquinones; Cell Survival; Disease Models, Animal; Drosophila; Enzyme Inhibitors; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Immunoblotting; Immunohistochemistry; Lactams, Macrocyclic; Lewy Bodies; Nerve Tissue Proteins; Neurons; Parkinson Disease; Proteins; Quinones; Synucleins; Temperature; Time Factors

We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.

Topics: alpha-Synuclein; Animals; Annexin A5; Apoptosis; Benzoquinones; Blotting, Western; Caspases; Cell Membrane; Cell Separation; Cysteine Proteinase Inhibitors; Cytochromes c; Flow Cytometry; Gene Deletion; Genotype; Glutathione; Green Fluorescent Proteins; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Mitochondria; Mutation; Nerve Tissue Proteins; Parkinson Disease; Plasmids; Quinones; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Synucleins; Time Factors

Geldanamycin (GA) is a naturally occurring benzoquinone ansamycin that induces heat shock protein 70 (Hsp70). GA has been shown to reduce alpha-synuclein induced neurotoxicity in a fly model of Parkinson's disease. We have previously shown that heat shock proteins can prevent alpha-synuclein aggregation and protect against alpha-synuclein induced toxicity in human H4 neuroglioma cells. Here, we hypothesize that GA treatment will reduce alpha-synuclein aggregation and prevent alpha-synuclein induced toxicity and we show that GA can induce Hsp70 in a time- and concentration-dependent manner in H4 cells. Pretreatment with 200nM GA 24h prior to transfection prevented alpha-synuclein aggregation and protected against toxicity. Treatment of cells with pre-existing inclusions with GA did not result in a reduction in the number of cells containing inclusions, suggesting that upregulation of Hsp70 is not sufficient to remove established inclusions. Similarly, Western blot analysis demonstrated that GA treatment could dramatically reduce both total alpha-synuclein and high molecular weight alpha-synuclein aggregates. Taken together, these data suggest that GA is effective in preventing alpha-synuclein aggregation and may represent a pharmacological intervention to therapeutically increase expression of molecular chaperone proteins to treat neurodegenerative diseases where aggregation is central to the pathogenesis.

Topics: alpha-Synuclein; Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; HSP70 Heat-Shock Proteins; Humans; Inclusion Bodies; Lactams, Macrocyclic; Nerve Tissue Proteins; Neurodegenerative Diseases; Quinones; Synucleins; Up-Regulation